This is a continuation of the "Enriched Artemia?" The Artemia in the photo are the larvae of Vietnamese strain 9 and a half hours post hatch. It took about 20 hours after hydration. The larvae can only feed endogenously when they grow to this stage. It usually take minimum of 2-3 hours for nutritional enrichment using a fortifier. Therefore, we must set the incubation of the dormant eggs for hatching at least one and a half day before the target feeding time
We use certain strain of brine shrimps (Artemia salina) to feed young larvae for our breeding marine ornamentals. We hatch the dormant eggs of Artemia. Since we know the hatched larvae are nutritionally insufficient for the ornamental larvae, we feed the Artemai larvae with certain fortifier. Are we, however, really feeding our larval ornamentals with enriched Artemia larvae?
The photo 1 is the empty shells of the dormant eggs of Artemia. The newly hatched Artemia has several stages (a-e in photo 2) to check. The stage indicated by "c" is called umbrella stage and the one indecated by "e" is the one we usually call hatched larva. This larva is at instar I stage. Showing in the photo 3 are the late Instar I and instar II stages. Check the difference between the two. IThe larava at Instar I does not have mouth nor anus. The Instar II has both open mouth and anus and can feed the food. It will take several hours from umbrella stage to Instar I and additional 10 hours may be necessary to further develop to Instar II stage. In the course of the nutritional enrichment of the larva Artemia we must not forget to consider this period of time or we may actually feeding our larval ornamentals the nutritionally deficient food.
I have two kind of rotifers. They are shown in the photo (below left). The bigger is Brachionus plicatilis and is called L type rotifer. The smaller is B. rotundiformis and called S type. The L-rotifer measured about 300 micron and the S-rotifer is more or less 200 micron. There is other type in S-rotifer, which is called SS (super small) type rotifer and measured less than 150 micron. The center photo shows L-rotifer with an egg. A rotifer gather the food particles through the current caused by their ciliary movement at the crown (photo of upper right).
When breding dottyback, Pseudochromis fridmani, collection of the hatched larvae is somewhat difficult matter. I found that the egg ball of the fish can be taken out prior to hatching and kept in the hatching bottle with vigorous aeration. At first I was afraid of certain damage to the hatcied larvae caused by bubbles hitting them so that I gave weak aeration and failed to hatch them out. The larvae during the first 2-3 days post hatching are tolerant about mechanical shock by the bubbles.
I had tested the effect of amount of aeration on the larval survival using the eggs and larvae of Humpback grouper, Cromileptes altivelis. Each of 200 eggs from the buoyant eggs collected from spawning tank was stocked in each of transparent circular tank (100 L) with varying aeration (0, 100, 500 and 1,000 ml/min). The set up was made in triplicate. The hatching rate was determined for each of the set up. As shown in the graph of upper right, the hatching rate does not differ much among the tanks with different intensity of aeration except the one without aeration.
The graph on the lower shows the effect of different strength of aeration on the larval survival of differnt developmental stages. During the yolk-sac stage from hatching to day 3, the larval survival was high with rather strong aeration. The larvae of the second stage, however, prefer weak aeration to the previous strong. Although not shown in the figure, the following stage of swimbladder inflationstage, require less strength in aeration. We, therefore, have to adjust aeration strength by the larval developmental stage. This explain the good result with storong aeration in the dottyback incubation.
Feeding mode of the fish (pseudochromis fridomani) changes as the larva debelops from one stage tothe next. At the time of hatching the larva depends its energy and nutrition on yolk reserves. The larva is at endogenous mode of feeding. Before the larva consumes its reserves, it starts feeding foods. The larva of this stage is at sucking mode of feeding. The larva preys on particulate foods whatever it encountered. When a prey is sighted the larva moves toward the food with its opened mouth. If prey is small enough to pass through the opened mouth and does not move away, the larva succedds in feeding. Since encounter rate is critical, food density oriented feeding is recommended for the larval rearing of this stage. As shown earlier, the larva at the notochord flexion stage is characterized by having teeth on jaws. The larva can now grasp aprey by its jaws so that the feeding of Artemia can be started at this stage. For a few days, however, a small amount of a newly hatched nauplii should be given as to acclimatize the larva to anew food. As the larva grows further, unpaired fins such as dorsal, caudal, and anal fins differentiate from fin fold so that the swimming function increase greatly. The larva can swim freely to search foods. The larva now is at selective feeding mode. From this time on, the feeding regimen should turn to rather frequency oriented than previous density oriented. By the time the larva completes metamorphosis, it tends to stay at bottomlayer, hide in darker portion of the tank. The larva positions itself at one place, poses for awhile till it sights a prey pass within its reach and ambush to attack the prey. The larva is at food chasing mode of feeding and develops cannibalism.
The fast growers among the others are metamorphosed into juveniles as early as day-35. The fins as well as the body are covered with blackdots as large as their eyes. Hence the fish is also called polka-dot grouper. Cannibalism develops. Squamation, scale formation, develops. The fish tends to settle to the bottom. With fanning mode of swimming the fish poises near the bottom. Then the fish ambush and attack the prey. They enter the food-chasing mode of feeding. Well before entering this mode, probably the time a third of fish metamorphoses into juvenile, a sieving to separate big fish from small must be done to avoid heavy mortality. The fish preys upon the fish slightly smaller than itself. The fishnormally can not ingest the prey. Both the cannibal and the prey would die. A week is enough for the number of fish to decline into half.
At day 35 when the fast growing larvae among the others reach the size of around 22-23 mm TL, they are almost completely juvenile stage. The metamorphosis, therefore, may have been started as early as in day-30 larvae and the fast growers measure around 18 mm TL. But the majority is still 12-13 mm TL at day 30. During this stage the elongated fin spines almost regress to normal size. Dark spots develop on body surface (Fig.). Pyloric caeca, which is known to improve fat digestion, appear between stomach and intestine. The shape of pectoral fins changes from the original circular form to adult form. Number of spines and rays of all the fins reaches to its adult form.
Vertebral column well develops so that the swimming ability increases further. The vertebral column, which replaces the notochord as the axial skeleton, consists of a series of rigid blocks separated by joints. The axial skeleton is believed to provide resilience to lateral bending and gives resistance to longitudinal compression.
At day 21 when the larvae reach around 7 mm TL, the notochord flexion is completed and unpaired fins are clearly separated from each other (arrow in the photo). The long spines have reached their maximum size and gradually decrease in size thereafter. Fin bases (pterigium catilages) for dorsal and anal fins appear. Fin fold disappears. The larvae start to swim against current caused by aeration. Number of teeth on lower jaw increase from previous stage through this stage. Upon sighting food, the larvae poised and taleJ-shaped posture prior to attacking the food.The larvae enter the selective feeding from the particulate feeding mode during this period so that feedinng regime should turn to frequency oriented from density oriented. Weaning to the artificial feed should be completed before the end of this stage.
By day 14 when the larvae reac around 5 mm TL, premaxillar teeth and detary teeth appear on jaws. There is one spine at inner preopercular and are two spines at outer preopercular. Their digestive tract has developed and coiled. (Stomach may have developed during this period.) Gill rakers start to develop. Shoulder girdle to support pectoral fin movement well develops during this stage. Notochord flexion starts and nearly completes followed by the unpaired fin differentiation during the period. The elongation of the fin spine proceeds further. The spines are folded during their burst swimming. The larvae are drifted helplessly with all three spines open when the larvae are not in healthy condition. Some of the larvae at this stage feed on harpacticoide copepods, which propagate in the tank. Although still under the particulate feeding stage, the larvae enter the grasp mode of feeding at the beginning of this stage so that Artemia feeding can be started. Since the weaning from rotifer to artificial diet must also start during this, it would be better if the artificial diet is given first in the morning and Artemia late in the afternoon. The larvae are still sensitive to any stress so that the stirring or disturbing the rearing water should be avoided. The tendency to aggregate seems to subside at around the middle of this stage.
At day 7 when the larvae reach around 3 mm TL, they are supposed to engulf air for their swim bladder dilation. Failure in air gulping may cause malformation of vertebral column. This usually is found only later during metamorphosisi stage by mass mortality of fish without dillalted swim bladder. Due to the formation of swim bladder (SW) the forgut is pushed down making V to Z shaped gut. Budss of the second dorsal fin spine (DS) and pelvic fin spines (VS) appear at the same time. These spines elongate fast and help the larvae to maintain balance. Aggregation toward the lighter portion of water becomes prominent. The larvae at this stage are very sensitive to any mechanical stress caused by water turbulence. Handling of the larvae or bottom cleaning, therforem should bi avoided.
At the night of day-2, usually 2 days after hatching (DAH), the mouth and anus are formed. A pair of pectoral fin buds appears several hours prior to mouth opening. Mouth width at the time of mouth opening is around 120 micron. The mouth width reaches 180-200 micron in around 10 hours. As shown in Fig.1, the day-3 larva when its mouth open has membranous pectoral fins and densely pigmented eyes with a small oil globule. With a pair of small pectoral fins and fin fold (primordial fin membrane) the larva can proceed forward by maintaining horizontal position. But they are merely drifted in the water and cannot move around by themselves. They can sight when a food happens to be in front of them, move toward the food so that the food passes through to mouth cavity. These larvae can ingest SS-type rotifer (Fig. 2). Fig 3 shows the rotifers (R) ingested. Although the anterior intestine is enlarged, the digestive tract is more or less straight from esophagus through anterior intestine. Only the posterior intestine (oe rctum) folds down to open at anus. They are in the mode of particulate feeding by sucking. Therefore the feeding system should be rather density oriented than feeding frequency oriented to increase the encouter rate of a larva to a food. When a rudimental swim bladder (Sb) appears at day 4 (Fig.4), the digestive tract is pushed down between stomach (St) and anterior intestine (Ain) and forms N-shape together with posterior intestine (Pin). Liver is clearly visible and rudimental oil globule (OG) still present. Even after the success of initial feeding and start of ezogenous feeding, the body length of the larva does not increase much (from 2.6 mm to 3.0 mm) compared to the rapid increase in mouth width (roughly from 300 to 500 micron).
The stage is between the time of hatching (or around 20 hours after spawning) and around 48 hours after hatching (HAH). The newly hatched larvae (photo) measure around 1.52 mm in total length (TL) and carry a large yolk and oil globule. As the larvae consume their reserves (yolk first followed by oil globule consumption: final absorption is around 4 days after hatch), they grow to ca. 2.58 mm TL just before mouth opening around two days after hatching. The anus situates rather posterior (preanal length to total length ratio is 0.68). The position of the anus moves anterior and situates more or less at the middle of body by the time of anus opening. Theyhave no pigments on eyes, no pectoral fins and no mouth during this period so that they can not sight the food, cannot move toward food and cannot take any food. The larvae are just suspended heads down in the water column with occasional jerk to recover their position as they sink gradually. This is the first critical stage suring the course of the grouper seed production. The larvae are often found dead at the water surface (floating death phenomenon).
In my writing of this blog, I often use some larvaldevelopmental stages. This staging was based on the morphological and behavioral development in the Humpback grouper, Cromileptes altivelis, of which I developed the seed production technique. I classified their developmental stage into 7 stages; Yolksac, Initial feeding, Swim bladder Inflation, Notocord flexion, Free swimming, Metamorphosis, and Juvenile. I am going to explain these developmental stages one at a day starting tomorrow.
The tomato clownfish has started feeding on Artemia nauplius yesterday. The larvae could already ingest newly hatched Aretemianauplius of Vietnamese starin. The larvae swimm well. This is understandable since they already are at the middle of notocord flexion stage and having proximal catilages of dorsal and anal fins in development.
I started to feed Artemia nauplius to the Day 7 larvae of the orchid dottyback yesterday. The nauplius is of the Vietnamese strain, which is slightly smaller than GSL starin at hatching. The larvae fed well on the Artemia.
The larvae of Tomato clownfish made surprise hatch out. Photo 1 shows the eggs and the male caring them at the back of the den. Photo 2 is the newly hatched larva. Photo 3, which is Day 5 larva of the orchid dottyback, is to compare with the newly hatched larva of the clownfish (photo4). They both are at the same developmental stage.
It seems that the heavy mortality experiencing last two days has ended. It is not known whether the result is because of reduced number of the larvae or the effect of the removal of earation. So far I lost almost one thousand larvae this time. The larvae are at Day 5 today and some of them may be able to take newly hatched Artemia of Vietnam strain. i, however would like to wait two more days before start feeding the Artemia. The reason to start feeding Artemia at Day 7 is to give all the larvae feed on Artemia so that I could avoid to ahve big size variation among the larvae.
The eggs of the Tomato clownfish are now loosing reddish color. I can even see the eyes in the egg shells shining. Since I failed to see the spawning, I am not sure of the spawning date. I have to be very careful about their change in coloration to gess the hatching date in order for assured larval collection.
I had big mortality of day 3 larvae of orchid dottyback yesterday. It seems therestill are so many dead larvae comming in the same batch. I suspect that the aeration may be causing the trouble to the larvae. I, therefore, remove the aeration to air-rift compartment. The rearing water is now only circulated by the incomming water through siphon.
The photos show the internal organs of the day 3 larva. In the photo 1 shows the liver (li), stomach (st), foregut (fg), and hider gut (hg). the foregut full of rotifers is shown in the photo 2. The last photo shows the end tip of the membranous fin of the larva. The tip is lobated as the anlagen of the hypural start appear.
I have harvested the juvenile orchid dottybacks grown up from 8th hatching yesterday. They were 66days old. I couted 65 of them. There was one malformed fish, which has curved vertebral column. So far I could only produce around 300 juveniles from 8 hatchings.
Tomato clownfish, Amphiprion frenatus, spawned yesterday. The parents were born December 2003. They, therefore, matured two years and four months after their birth. I would like to try to produce F2 from these eggs. I, however, have to find the sppace for the larval rearing of these fish, since almost all of my available containers are now occupied by the orchid dottybacks.